The Edman degradation is a well established method for the sequential degradation of protein. Three reactions are required to remove the amino-terminal amino acid and convert it to a form which is suitable for analysis. The first reaction (coupling) modifies the amino terminus by the addition of phenylisothiocyanate (PITC) to the amino group. This is usually a base-catalyzed reaction. The resulting phenylthiocarbamyl (PTC) protein is then treated with an anhydrous acid in a second reaction (cleavage) which allows the sulfur from the PTC group to react with the first carbonyl carbon in the protein chain. This cyclization reaction results in the removal of the first amino acid as an anilinothiozolinone (ATZ) derivative and leaves the next amino acid in the protein exposed for the next round of PITC coupling. In a third reaction (conversion), the ATZ amino acid is converted to a phenylthiohydantoin (PTH) amino acid in aqueous acid. The PTH is more stable than the ATZ and can be easily analyzed. This process may be continued until the limitations of the chemistry or the sample preclude further analysis.
Generally, the phenylthiohydantoin (PTH) amino acid has been considered to be the end product of the Edman degradation since Edman first described this process for the automated sequential analysis of proteins (ref.1). The PTH is a relatively stable form of the amino acid and is readily generated from the products of the Edman acid cleavage step by treatment with aqueous acid. This conversion reaction provides an easy way to obtain a single PTH amino acid derivative from the mixture of ATZ, PTC and PTH amino acids which are present after the cleavage step. It should be noted that while the theoretical product of the cleavage reaction is the ATZ, in actual practice significant amounts of the PTC and PTH derivatives are present due to the partial conversion of the ATZ in the acidic post-cleavage environment. Other factors, such as the presence of reducing agents in the sequencing chemicals, also influence the final proportion of the amino acid derivatives. The extent of this conversion also depends somewhat on the specific amino acid, with the aspartic acid ATZ derivative exhibiting the greatest tendency to form PTH under the anhydrous acidic cleaving conditions.
It is possible to analyze the PTH amino acids in the low picomole range using high-performance liquid chromatography (HPLC) and ultraviolet (uv) absorption techniques. While this level of performance is acceptable for most applications, many naturally-occuring peptides and proteins of interest are obtainable only in extremely low (sub picomolar) amounts and cannot be analyzed with current sequencing methods because of limitations in uv detection sensitivity.
New developments in protein micro-preparation techniques have allowed minute quantities of purified protein to be prepared for sequencing. For example, the recently developed technique of isolating protein by gel electrophoresis followed by transfer to sequencer-compatible membranes (ref.2) has allowed sub-picomole amounts of sample to be prepared in purified form However, protein bands on the membrane, which are visible by conventional staining techniques, are often at too low a concentration for successful sequence determination. The inventor herein believes that the major factor preventing analysis at these low levels is the method of PTH amino acid identification, not inherent limitations in the Edman chemistry or instrumentation. It is, therefore, desirable to leave the sequencing chemistry and instrumentation intact and explore ways to alter the end products in ways which will increase their sensitivity of detection.
Inman and Appella (ref.3) described a method for visualizing the end products of the Edman reaction by foregoing the aqueous acid conversion step and reacting the ATZ amino acids present in the post-cleavage mixture with methylamine. These products, phenylthiocarbamyl amino acid methylamides (PTMA amino acids), absorb in the ultraviolet range so no significant enhancement in sensitivity is realized over PTH analysis. However, their report is of interest since it describes a method by which the ATZ amino acids present after cleavage may react with primary amines. Tsugita et al (ref.4) and Horn et al (ref.5) have taken this approach and applied it to methods which offer more sensitivity of detection. They have shown that the ATZ amino acid is reactive with radioactive and fluorogenic primary amines (Tsugita) or fluorogenic alcohols (Horn). These compounds are easily detected at levels far below those possible with ultraviolet absorption.
While both the Tsugita and Horn methods offer the possibility for successful sequence analysis below the picomole level, they also suffer from several shortcomings. For example, while the theoretical end product of the Edman degradation after acid cleavage is the ATZ amino acid, there is always some PTC and PTH amino acid present as well. The PTC and PTH amino acids, unlike the ATZ amino acids, do not react with the aforementioned primary amines or alcohols to yield the desired detectable species, thus lowering the effective sensitivity of the method. In the case of some of the amino acids, almost all of the product may be PTC and/or PTH due to acid conversion of these amino acids during the cleavage step of the degradation (see above). Furthermore, it is common practice to include a reducing agent in the Edman chemicals to help scavenge trace amounts of oxygen or peroxides which will poison the degradation chemistry. The adverse effects of these oxidizing contaminants are proportionately larger as smaller amounts of protein are analyzed. This is easily observed as a sharp decrease in the repetitive efficiency of the Edman degradation as lower and lower amounts of sample are sequenced. Unfortunately, the presence of a reducing agent, while protecting the degradation chemistry, greatly shifts the proportion of post-cleavage products toward the PTH, leaving almost no ATZ available for the sensitivity-enhancing reaction (see FIG. 1). This effect seems to be amplified, again, as lower and lower amounts of sample are analyzed.
Also, there is compelling evidence for the existence of more than one form of ATZ amino acid present in the post-cleavage mixture (see FIG. 2a). One of these putative tautomers is not reactive with primary amines or alcohols. A typical post-cleavage mixture may contain a significant amount of this non-reactive form. Further treatment of the mixture with neat trifluoroacetic acid shifts the ratio to the reactive form but also increases the proportion of PTC and PTH in the mixture (see FIG. 2b). All of these effects collaborate to greatly decrease the amount of ATZ available for the sensitivity-enhancement reaction.
In published sequence data obtained by using enhancement chemistry which acts directly on the post-cleavage Edman products, all of these effects are clearly evident, especially with regard to the levels of the enhanced products of aspartate, glutamate, asparagine and glutamine which appear to be absent entirely (ref.4). This effect is illustrated in FIG. 3. The present invention seeks to overcome these adverse effects by converting the post-cleavage mixture of PTH, PTC and ATZ amino acids to a homogeneous reactive ATZ amino acid, and also to allow the use of reducing agents in the Edman sequencing chemicals to preserve the efficiency of the degradation without decreasing the efficiency of the sensitivity-enhancement chemistry.